Accumulation of flavonoids in soybean under effects of cyanobacterial crude extract and aphid infestation

Journal of Science and Technology 54 (2C) (2016) 271-277 ACCUMULATION OF FLAVONOIDS IN SOYBEAN UNDER EFFECTS OF CYANOBACTERIAL CRUDE EXTRACT AND APHID INFESTATION Mai Van Chung1, *, Doan Manh Dung2, Do Ngoc Dai3 1Vinh University, 182 Le Duan, Vinh city, Nghe An province 2Hue University of Science, 77Nguyen Hue, Hue city, Thua Thien Hue province 3Nghe An College of Economics, 51 Ly Tu Trong, Vinh city, Nghe An province *Email: chung.uni@gmail.com Received: 15 June 2016; Accepted for publication: 15 October 2016 ABSTRACT The differential accumulation of endogenous flavonoids was recorded in soybean (G. max (L.) Merr. cv. “Nam Dan”) under infestation of cowpea aphid (Aphis craccivora Koch) and/or treatment of the crude extract of cyanobacterium strain Nostoc calcicola HN9-1a. During A. craccivora infestation, flavonoids in the aphid-infested leaves were strongly induced to high contents, which were 1.84–2.21 - fold higher than that observed in control. Those bioactive substances were suppressed by the single treatment of N. calcicola crude extract, however, were enhanced by the cross-talk interactions of N. calcicola HN9-1a and A. craccivora. Flavonoids in the double factors-effected soybean leaves were remarkably increased to high level since 24 hours after cowpea aphid attack, having by 2.99 - 3.06-fold higher than that in control. It was the important evidences to suggest that flavonoids may function in the defense mechanism of soybean “Nam Dan” against A. craccivora; and N. cacicola HN9-1a crude extract improved the accumulation of flavonoids in soybean response to cowpea aphid infestation. Keywords: flavonoids, cyanobacteria, soybean “Nam Dan”, cowpea aphid. 1. INTRODUCTION Cyanobacteria are known to produce various kinds of bioactive compounds that affect many physiological processes within living cells. Species belonging to genus Nostoc are regarded as good candidate for producing secondary substances that influence the growth of plants [1]. A large number of properties of Nostoc spp. have been identified as cytotoxic, antifungal, antibacterial, antiviral, immunosuppressive, enzyme inhibiting activities in the effect on higher plants [2]. Extract of some Nostoc species induces oxidative stress in plant cells by reactive oxygen species (ROS) productions resulting in lipid peroxidation and massive cell death as well as activating enzymatic antioxidants [3]. In our recent publication mentioned about the effect of strain N. calcicola HN9 on the antioxidant system of soybean “Nam Dan” (Glycine max Mai Van Chung, Doan Manh Dung, Do Ngoc Dai 272 (L.) Merr.), we revealed that, N. calcicola HN9 in death phase resulted the oxidative stress and enhanced activity of antioxidant enzymes such as superoxide dismutases (SOD), catalase (CAT), ascorbate peroxidases (APX) and polyphenol oxidase (PPO) in soybean leaves [4]. Effects of Nostoc extract on plant defense mechanism, however, remain poorly understood. A number of chemotypes of flavonoids, including flavones, isoflavonoids, flavonoid-glycosides, have been known to protect crops against insects herbivory [5]. In legumes, these compounds reduce plants’ nutritive value, decrease digestibility of insects [6], or function as preformed or inducible anti-insecticidal properties [7], evenly act as toxins to pests [8]. Flavonoids also scavenge the free radicals, including ROS productions, and control their formation in living cells, therefore, reduce the oxidative damages [9]. To date, the available information of the insecticidal properties towards aphis of flavonoids in soybean plants is limited. In this study, we focused on the biochemical responses of Glycine max (L.) Merr. cv. “Nam Dan” to cowpea aphid-Aphis craccivora Koch (Hemiptera: Aphididae) concerning the expression of endogenous flavonoids. To partly assess the role of cyanobacteria on biosynthesis of flavonoids in soybean defense mechanism, we analyzed the changes of flavonoids’ content in G. max cv. “Nam Dan” leaves treated by the crude extract of strain Nostoc calcicola HN9-1a, which is hypothesized as an elicitor to soybean responses to aphid herbivores. 2. MATERIALS AND METHODS 2.1. Materials 2.1.1. Plant Plant used in the experiments is cultivar “Nam Dan” of soybean (Glycine max (L.) Merr.). Soybean seeds have been exclusively provided by Nam Dan Agricultural Extension Center (Vietnam). Soybean plants were cultured in 20-cm-diameter plastic pots containing Hoagland medium placed in the phytotron with temperature of 23 - 25o C, related humidity of 70 – 75 %, light intensity of 110 - 130 μM photons.m-2.s-1 and light period of 12 light /12 dark hours in the Plant physiology lab, Vinh University. 2.1.2. Aphid Cowpea aphid (Aphis craccivora Koch) is cultured and supported by Department of Applied Entomology (Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology). 2.1.3. Cyanobacteria culture and extraction The cyanobacterium strain, Nostoc calcicola HN9-1a, was collected from the rice field in Hung Nguyen district (Nghe An province) and isolated and cultured in the Phycology lab (Vinh University). Nostoc cells were cultured in BG11 medium, pH 6.5 at temperature of 32 ± 2°C, under daylight fluorescent lamps, light period of 14 light/10 dark [3]. The N. calcicola HN9-1a biomass harvested in the stationary phase were centrifuged at 5,000 × g for 20 min and subsequently dried at 50 oC for 72 hours. The dried cells were ground to powder and extracted with 80 % methanol for 24 hours. The solution was centrifuged by 10,000 × g in 15 min to collect supernatant that was evaporated to obtain a crude brown gum [3]. Accumulation of flavonoids in soybean under effect of 273 2.2. Experiment The cyanobacterial gum was dissolved in the distilled water at 0.00, 0.03 % and 0.05 %. These solutions were separately sprayed in leaves of soybean “Nam Dan” when plants were in stage V3 (three fully unrolled trifoliolates). After spraying 24 hours, each soybean plant was treated by 10, 20 or 30 wingless adults of A. craccivora. The control was soybean plants without aphid infestation. All variants were separately put in glass boxes (50 cm × 50 cm × 50 cm) covered by nylon gauze and placed in the phytotron with the environmental factors such as temperature, relative humidity, light intensity and light period controlled strictly. Leaves of soybean plants were collected after 0, 24, 48, 72 and 96 hours post-infestation (hpi) of cowpea aphid. After all aphid individuals were carefully removed, leaves were weighed, frozen in nitrogen liquid and kept at -70 oC for subsequent analyses of flavonoids. 2.3. Analysis 2.3.1. Chemicals All analytical chemicals were purchased from Singapore supplier of Sigma-Aldrich (USA). 2.3.2. Analysis of total flavonoids in soybean leaves Total of 0.50 g frozen soybean leaves was extracted with 10 mL of 99.5 % ethanol under 200 rpm shaking for 24 hours. After filtration, the filtrate was adjusted to 10 mL with 80 % ethanol and centrifuged at 10,000 × g for 10 min at 4 oC, the supernatant was collected and the precipitate was then extracted with 5 mL of 80 % ethanol twice. Finally, the supernatant was combined with previous supernatant and adjusted to 20 mL with 80 % ethanol for analyses [10]. Content of total flavonoids in soybean leaves extract was determined by the aluminum chloride colorimetric method [10]. A mixture of 200 µL extract and 150 µL of sodium nitrite (NaNO2 5 %, w/v), was firstly incubated for 6 min at room temperature. Next, 150 µL of aluminium chloride hexahydrate (AlCl3.6H2O 10 %, w/v) was added and incubated for 6 min at room temperature, then 1,000 µL NaOH (10 %, w/v) solution was added; total of 1,500 µL mixture was incubated at room temperature for 25 min. The absorbance was measured at λ = 510 nm in the spectrophotometer UV-Vis CARY 60 (Agilent, USA) connected with a computer installed the data analytical software Agilent Cary WinUV 5.0. The calibration curve was established using quercetin dissolved in 80 % ethanol and then diluted to 25, 50, 100 and 200 µg.mL-1 as the standards. Total flavonoids was calculated from the calibration curve y = 0.186x - 0.79 (the correlation coefficient R2 = 0.9965) and were expressed in microgram quercetin equivalent per gram dry matter (μg QE.g-1 dw). 2.3.4. Statistical analysis All analyses were performed in at least three replicates. Analysis of variance (ANOVA) was applied to verify whether means from independent experiments within each given variant were significant at level P < 0.05. Data shown in the figures are means and standard errors (s.e.). Mai Van Chung, Doan Manh Dung, Do Ngoc Dai 274 3. RESULTS AND DISCUSSION 3.1. Infestation of cow pea aphid accumulated flavonoids in soybean leaves Infestation of A. craccivora accumulated generation of flavonoids in G. max (L.) Merr. cv. “Nam Dan”. In aphid-infested leaves, levels of flavonoids were immediately increased and were always higher than in the control during experiment (Fig. 1). The highest content of flavonoids was 149.32 μg QE.g-1 dw in 30 aphid-infested leaves at 72 hpi, which was by 2.21- and 1.84- fold higher than at the beginning of experiments and in control plants, respectively. The high content of in flavonoids’ in soybean “Nam Dan” leaves was resulted from the high infestation intensity from cowpea aphid. The significant differences between flavonoids levels in aphid- infested variants and control were recorded from 48-96 hpi (P < 0.05). Figure 1. Generation of total flavonoids in leaves of G. max cv. “Nam Dan” control and A. craccivora infested leaves without treating Cyanobacteria. Previous studies suggested a positive relationship between flavonoid content and the resistance/susceptibility characteristics against aphids of leguminous plants [11], whereby, the resistant lines showed the high accumulation of flavonoid under aphid effects. A strong generation of flavonoids in G. max cv. “Nam Dan” after A. craccivora infestation is an important evidence to suggest that, this cultivar maybe the resistant cultivar to cowpea aphid. 3.2. Cyanobacterial crude extract suppressed biosynthesis of flavonoids in soybean leaves Figure 2. Effect of N. calcicola HN9-1a crude extract to content of flavonoids in G. max cv. “Nam Dan” without infestation of cowpea aphid. Accumulation of flavonoids in soybean under effect of 275 Crude extract of N. calcicola HN9-1a expressed to suppress biosynthesis of flavonoids in leaves of soybean “Nam Dan” (Fig. 2). Both two concentrations of extract such as 0.03 % and 0.05 % trended to reduce generation of flavonoids. Content of flavonoids in leaves treated by N. calcicola HN9-1a continuously decreased since 24 hpi and were significantly lower than in control within 48-96 hpi (P < 0.05). The lowest content of flavonoids recorded in soybean leaves treated by 0.05 % extract at 96 hpi was 41.75 μg QE.g-1 dw, having by 54.23 % in comparing with control at the same point of time. Flavonoids are often generated strongly when plants faced to stresses [9]. However, lack of information from available documents regarded the effected mechanism of cyanobacteria and their extract on biosynthesis of flavonoids in higher plants. This important aspect of the Cyanobacteria-soybean interaction should be clarified in the prospective studies. 3.3. Cyanobacterial crude extract improved the accumulation of flavonoids in soybean leaves infested by cowpea aphid The cyanobacterial crude extract of N. calcicola HN9-1a reduced flavonoids’ levels in leaves of G. max cv. “Nam Dan” (Fig. 2), but it seems to improve the accumulation of flavonoids in soybean leaves after aphid infestation. Content of flavonoids in all aphid-infested soybean plants treated by N. cacicola extract was enhanced and remarkably increased to high levels within 24-96 hpi, which were significant higher than in control (P < 0.05) (Fig. 3). A b Figure 3. Content of flavonoids in leaves of G. max cv. “Nam Dan” under infestation of A. craccivora and treatment of N. calcicola HN9-1a extract solution 0.03 % (a) and 0.05 % (b). The 0.03 % N. calcicola HN9-1a extract induced flavonoids in the infested soybean leaves strongly reached to peak within 48-72 hpi. The highest content of flavonoids obtained in the 20 aphid-infested leaves at 48 hpi was 207.79 μg QE.g-1 dw, having by 2.99- and 3.06- fold higher than that observed in beginning and in control, respectively (Fig. 3a). Denoting the changing in content similar to effect of 0.03 % N. calcicola extract, however, flavonoids in soybean leaves treated by 0.05 % concentration reached to maximum levels later, was within 72 hpi, and the highest level was resulted by infestation of 30 aphid individuals; whereas that in the 0.03 %-treated variant was 20 aphids (Fig. 3b). Mai Van Chung, Doan Manh Dung, Do Ngoc Dai 276 In summarizing, generation of flavonoids in soybean “Nam Dan” under the cross-talk interaction of N. calcicola HN9-1a extract and A. craccivora infestation was early and remarkably accumulated. Their content was much higher than that observed in variants infested by cowpea aphid only. The crude extract of N. calcicola HN9-1a may elevate the accumulation of flavonoids in leaves of soybean “Nam Dan” under infestation of A. craccivora. 4. CONCLUSION The high efficiency of flavonoids in regulation of ROS products has been known to constitute important lines of defense mechanism protecting plants against oxidative damages caused by infestation of aphids [4, 8]. The accumulated levels of flavonoids in A. craccivora- infested leaves of G. max cv. “Nam Dan” acted as a potential defense against cowpea aphid, while the crude extract of N. calcicola HN9-1a additionally enhanced the accumulation of those antioxidants; under effect of cyanobacteria, flavonoids in the aphid-infested leaves was remarkably increased to high level. It was suggested that flavonoids might be a vital element in the defense mechanism of soybean “Nam Dan”, and N. cacicola HN9-1a crude extract seemed to improve function of flavonoids in soybean response to infestation of cowpea aphid. Acknowledgement:.This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number: 106-NN.03-2014.22. REFERENCES 1. Hirata K., Yoshitomi S., Dwi S., Iwabe O., Mahakhant A., Plochai J., and Miyamoto K. - Bioactivities of Nostocine-a produced by a freshwater cyanobacterium Nostoc spongiaeforme TISTR 8169, J. Biosci. Bioeng. 95 (2003) 512-517. 2. Nowruzi B., Khavari N. R. A., Sivonen K., Kazemi B., Najafi F., and Nejadsattari T. - Identification and toxigenic potential of a Nostoc sp., Algae 27 (2012) 303-313. 3. Sukkhaeng S., Sanevas N., and Suwanwong S. - Nostoc sp. extract induces oxidative stress mediated root cell destruction in Mimosa pigra L., Botanical Studies 56 (2015) 3 DOI 10.1186/s40529-014-0081-3. 4. Mai V. C., Mai T. T. H., and Dinh T. T. G. - Effect of Cyanobacteria Nostoc calcicola on antioxidant system in soybean. Journal of Science and Technology (Vietnam), 54(2) (2016): 238-248. 5. Simmonds M. 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Lattanzio V., Arpaia S., Cardinali A., Di Venere D., and Linsalata V. - Role of endogenous flavonoids in resistance mechanism of Vigna to aphids, J. Agric. Food Chem. 48 (2000) 5316-5320. TÓM TẮT SINH TỔNG HỢP CỦA FLAVONOID Ở ĐẬU TƯƠNG DƯỚI TÁC ĐỘNG CỦA DỊCH CHIẾT VI KHUẨN LAM VÀ RỆP HẠI Mai Văn Chung1, *, Đoàn Mạnh Dũng2, Đỗ Ngọc Đài3 1Trường Đại học Vinh, 182 Lê Duẩn, Tp. Vinh, Nghệ An 2Trường Đại học Khoa học-Đại học Huế, 77 Nguyễn Huệ, Tp. Huế, Thừa Thiên Huế 3Trường Đại học Kinh tế Nghệ An, 51 Lý Tự Trọng, Tp. Vinh, Nghệ An *Email: chung.uni@gmail.com Dịch chiết vi khuẩn lam Nostoc calcicola HN9-1a và rệp muội đen (Aphis craccivora Koch) đã cảm ứng khác nhau đối với sinh tổng hợp flavonoid trong lá đậu tương (Glycine max (L.) Merr. cv. “Nam Đàn”). Dưới tác động của rệp muội đen, flavonoid trong lá đậu tương “Nam Đàn” đã cảm ứng tăng cao 1,84 - 2,21 lần so với đối chứng, và lượng flavonoid sinh ra tỷ lệ thuận với mức độ tác động của rệp. Tác động riêng của dịch chiết vi khuẩn lam N. calcicola HN9-1a không cảm ứng đối với flavonoids nhưng trong sự tương tác với rệp hại, đã kích thích tổng hợp chất chống ôxy hóa này mạnh hơn 2,99 - 3,06 lần so với đối chứng. Cảm ứng sinh tổng hợp flavonoid là một phản ứng đáp trả của cây đậu tương “Nam Đàn” đối với sự phá hại của rệp muội đen; và dịch chiết N. calcicola HN9-1a đã có tác dụng tăng cường vai trò của flavonoid trong cơ chế tự bảo vệ của giống đậu tương này đối với A. craccivora. Từ khóa: vi khuẩn lam, đậu tương “Nam Đàn”, rệp muội đen, flavonoid.